To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heatshocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.

Human mesenchymal stromal cells (hMSCs) are capable of limited self-renewal and multilineage differentiation in vitro. Several studies have demonstrated that microRNAs (miRNAs, miRs), post-transcriptional modifiers of mRNA stability and protein translation, play crucial roles in the regulation of these complex processes. To gain knowledge regarding the role of miRNAs in human adipocyte differentiation, we examined the miRNA expression profile of the immortalized human bone marrow-derived stromal cell line hMSC-Tert20. Such a model system has the advantage of a reproducible and consistent phenotype while maintaining important properties of the primary donor cells, including the potential to differentiate to adipocytes, osteoblasts, and chondrocytes. We identified 12 miRNAs that were differentially expressed during adipogenesis, of which several have been previously shown to play important roles in adipocyte biology. Among these, the expression of miRNA-155, miRNA-221, and miRNA-222 decreased during the adipogenic program of both immortalized and primary hMSCs, suggesting that they act as negative regulators of differentiation. Interestingly, ectopic expression of the miRNAs significantly inhibited adipogenesis and repressed induction of the master regulators PPARγ and CCAAT/enhancer-binding protein alpha. Our study provides the first experimental evidence that miRNA-155, miRNA-221, and miRNA-222 have an important function in human adipocyte differentiation, and that their downregulation is necessary to relieve the repression of genes crucial for this process.

Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and highresolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.

Circadian clocks have been shown to operate developmentally in mouse and human hematopoietic stem and progenitor cells in vivo, but little is known about their possible oscillations in vitro. Here, we show that repeated circadian oscillations could be induced in both cultured bone marrow-derived mesenchymal- and adipose-derived stem cells (MSCs and ASCs, respectively) by serum shock. In particular, the novel finding of rhythmic clock gene expression induced by cAMP analogs showed similarities as well as differences to serum-induced oscillations. Rhythmic PER1 expression was found in serum-shocked MSCs, suggesting the phosphorylation status of PER1 is important for its activity in circadian rhythms. Furthermore, immunofluoresent staining showed that the localization of PER1 was dependent on the level of PER1 expression. These inducible self-sustained circadian clocks in primary cultures of human MSCs in vitro with rhythmic changes in expression levels, phosphorylation, and localization of clock protein, PER1, may be of importance for maintaining the induced oscillations in stem cells. Therefore, the established cell models described here appear to be valuable for studying the molecular mechanism driving and coordinating the circadian network between stem and stromal cells.

BackgroundIn vitro studies of osteoblasts traditionally use Alizarin Red as a golden standard for the detection and quantification of mineralization, which is a marker of osteoblast differentiation. However, this method presents a number of drawbacks, including the need to fix cells, which prevents additional measurements. Years ago, Calcein Green was proposed as an alternative to Alizarin Red, with the advantage to be directly detectable in live cells. However, the protocol was still time-consuming, and it never managed to replace Alizarin Red. Now, with more efficient imaging systems, we present a protocol using Calcein Green which provides significant advantages.ResultsThe osteoblast mineralization was efficiently detected and accurately quantified in real time at any desired time point across the entire differentiation period, with a minimum time expenditure.ConclusionsThe combination of Calcein Green and the real-time imaging station IncuCyte ZOOM can efficiently replace the Alizarin Red method, and allows very accurate and time-saving assessment of the level and the dynamics of matrix mineralization.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0079-4) contains supplementary material, which is available to authorized users.